Yearly, millions of people die, or are seriously debilitated, from malaria, dengue fever, yellow fever and several forms of viral encephalitis. Hematophageous mosquitoes transmit the causative agents of these diseases [1, 2]. As global warming, and migration of human and mosquito populations increases, dengue fever may spread to dengue-free regions of the United States [3-8]. Current methods to control these diseases emphasize immunization, chemical prophylaxis, control of the insect vectors, and reduced contact with insect vectors. They are useful, but the diseases remain significant problems, especially in underdeveloped tropical countries [9, 10]. Although widely used, little is known concerning the molecular mechanisms by which chemical vector control agents work, leaving limited options to modify or improve these methods of control. New initiatives targeting mosquito biology are in development. One initiative is the development of transgenic mosquitoes having a reduced ability to transmit the disease agents [11-18]. In pursuit of this goal, molecular-genetic techniques have been applied to mosquito research. These include the sequencing of the Anopheles gambiae[19], Aedes aegypti [20] and Culex pipiens genomes[21], developing techniques to produce stable transgenic mosquitoes [22-32], the use of RNAi techniques [16, 33-37] and development of genetic drive systems [24, 38-41]. Additionally, molecular-genetic techniques may reveal molecular mechanisms by which chemical mosquito control agents work. Along this line, our long term goal is to understand the molecular- genetic mechanisms that control mosquito larval midgut growth and metamorphosis in order to identify processes that can be exploited to better control populations of hematophageous, disease carrying mosquitoes. The information obtained may result in the design of more specific and bio-rational larvicidal chemicals, and in the design of transgenic mosquitoes in which growth and metamorphic processes are altered so that adult population densities, or fecundity, are reduced. The central hypothesis driving this proposal is the genes methoprene tolerant (met) and broad (br) are central to the transcription factor cascade that controls metamorphosis, and in the pathway by which juvenile hormone analogues, widely used in commercial larvicides, interfere with metamorphosis. Proposed here are genetic tests of the central hypothesis. We use mosquitoes because the information we discover will likely be directly applicable to the control of mosquito populations. This approach is now possible because we have developed RNAi techniques for use in mosquito larvae, giving us a unique opportunity to genetically examine the role that various genes play in mosquito metamorphosis. To further the genetic analysis of our central hypothesis, and in investigations of other factors controlling mosquito growth and metamorphosis, we propose to develop in vivo transient transfection of mosquito larvae. Effective control of mosquito borne diseases will require an integrated approach [42] including transgenic mosquitoes, chemical control, avoidance of mosquitoes and immunization. ) PUBLIC HEALTH RELEVANCE: Mosquitoes are not just pests but can transmit the agents that cause deadly and seriously debilitating diseases such as malaria, dengue fever and westnile encephalitis. The long term goal of this research is to understand the molecular mechanisms that control mosquito larval growth and metamorphosis in order to identify potential targets that can be exploited to better control the number of blood-sucking, disease carrying mosquitoes. We propose to test, by knockdown of gene expression, the hypothesis that the genes broad and methoprene tolerant are central to mosquito development, and are in the pathway by which some insecticides block mosquito development.